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Cytogenetics

Postwash

The post-hybridization wash, or “stringency wash,” is the cleanup phase of the FISH procedure. After the overnight hybridization, the slide is covered in fluorescent probe. While some of this probe is bound tightly to its specific DNA target (e.g., the HER2 gene), a significant amount is bound loosely to imperfect matches (non-specific sequences), the cytoplasm, or the glass slide itself. The purpose of the postwash is to strip away this “noise” while leaving the specific “signal” intact. This discrimination is achieved by manipulating the chemical concept of Stringency

The Concept of Stringency

Stringency describes the conditions that determine how perfectly two strands of DNA must match to remain bound together

  • High Stringency: Only perfect, 100% complementary matches survive. Weak, mismatched bonds are broken
  • Low Stringency: Imperfect matches (e.g., 80% homology) can remain bound

In clinical FISH, we require High Stringency to ensure that a green signal actually represents the target gene and not a similar sequence elsewhere in the genome. Stringency is controlled by two variables: Temperature and Salt Concentration

  • Temperature: Heat adds kinetic energy to the DNA strands
    • Higher Temperature = Higher Stringency. Heat shakes the molecules apart. Only the strongest bonds (perfect matches with many hydrogen bonds) can withstand the shaking
    • Standard: Typically \(72^\circ\text{C}\) (\(\pm 1^\circ\text{C}\)): for rapid washes or \(45^\circ\text{C}\)–\(50^\circ\text{C}\) for Formamide-based washes
  • Salt Concentration (SSC Buffer)
    • DNA is negatively charged (phosphate backbone). Salt (Sodium Chloride) creates a cloud of positive ions that shields these negative charges, allowing strands to stick together easily
    • Lower Salt = Higher Stringency. By removing the salt (using a dilute buffer like 0.4x SSC), the negative charges repel each other. Only perfectly matched strands stay together
    • Standard: 2x SSC: (Standard Saline Citrate) or 0.4x SSC

The Wash Protocol

  1. Removal of Coverslip: The rubber cement is carefully peeled off, and the coverslip is gently soaked off in a room-temperature buffer. Tearing the coverslip off can rip the cells off the glass
  2. The Stringency Bath: The slide is immersed in the pre-heated wash buffer (e.g., 0.4x SSC/0.3% NP-40 at \(73^\circ\text{C}\)) for exactly 2 minutes
    • Timing: Critical. 1 minute is insufficient to remove background; 5 minutes might wash away the specific signal
    • Agitation: Gentle agitation ensures fresh buffer contacts the slide surface
  3. The Rinse: The slide is moved to a room-temperature buffer to cool down and remove the detergent
  4. Drying: The slide is air-dried in the dark (fluorescent dyes are light-sensitive) before applying DAPI counterstain

Troubleshooting the Wash

The quality of the final image is often determined here

  • High Background (Green Haze)
    • Diagnosis: Stringency was too Low.: The wash didn’t remove the excess probe
    • Cause: Wash buffer was too cold (checking the thermometer is vital) or salt concentration was too high
  • Weak/No Signal
    • Diagnosis: Stringency was too High.: The wash stripped the probe off the target gene
    • Cause: Wash buffer was too hot (\(>74^\circ\text{C}\)) or the slide was left in the bath too long
    • Note: This can also be caused by insufficient probe initially, but if the DAPI is good and signal is zero, over-washing is a prime suspect

Detergents (NP-40 / Tween-20)

Wash buffers almost always contain a non-ionic detergent like NP-40 (Nonidet P-40) or Tween-20

  • Function: These surfactants reduce surface tension. They help the buffer penetrate the slide surface and, crucially, help “scrub” the lipophilic fluorescent dyes off the glass and cytoplasm, reducing non-specific background fluorescence